Overactive bladder (OAB), often associated with detrusor overactivity (DO), is a debilitating pathological condition affecting more than 34 million Americans, at an annual cost of more than $65 billion in the US alone. Current OAB therapies are limited in efficacy and relief. Novel therapeutic approaches are urgently needed, especially those that directly target detrusor smooth muscle (DSM), in order to facilitate DSM relaxation during bladder filling and urinary storage. We are the first to identify functional Transient Receptor Potential (TRP) melastatin-4 (TRPM4) channels in human DSM and we have shown that TRPM4 channel expression is much higher in DSM as compared to the vasculature. This project will examine the novel concept that the Ca2+- activated TRPM4 channels have a key role in human DSM excitability and contractility and that inhibition of TRPM4 channels decreases DSM contractility. Our central hypothesis is that TRPM4 channels play critical roles in human DSM physiology and the etiology of DO/OAB, and therefore represent a novel therapeutic target for DO/OAB treatment. Our strategy is to use a multi-level experimental approach at molecular, cellular, tissue, and whole organism levels. We have the unique advantage of regularly using clinically-characterized human DSM tissues from open bladder surgeries on control (non-OAB) and OAB patients. Aim 1 will use multiple state-of-the-art approaches including single-cell RT-PCR, qPCR, Western blot, in situ proximity ligation assay, confocal immunohistochemical and immunocytochemical analyses of DSM whole tissue and single cells to identify the TRPM4 channel expression and localization in human DSM cells from control and OAB patients. Aim 2 will elucidate the role of TRPM4 channels in the excitability of freshly- isolated (not cultured) native human DSM cells from control and OAB patients and their functional link with IP3 receptors. This aim will use advanced electrophysiological approaches, live-cell Ca2+ imaging, and the novel selective TRPM4 channel inhibitor, 9-phenanthrol. Aim 3 will elucidate the functional roles of TRPM4 channels in myogenic and nerve-evoked contractions of human DSM tissues from control and OAB patients, and in vivo in mice. We propose that TRPM4 channels are novel targets for DSM dysfunction and their pharmacological modulation offers a previously unexplored mechanism for treating bladder disorders. Targeting TRPM4 channels with selective inhibitors has the potential to reduce DO and alleviate OAB. Therefore, this investigation has important clinical significance. The highly innovative nature of this translational research is strengthened by the collaboration between basic scientists and urologists, allowing the linking of molecular (Aim 1), cellular (Aim 2), tissue, and in vivo (Aim 3 findings. Upon completion of this project, the regulation of human DSM by TRPM4 channels under physiological and pathophysiological conditions will be revealed. The knowledge gained from this project will advance our understanding of the mechanisms regulating human DSM function and dysfunction and validate TRPM4 channels as a novel therapeutic modality for OAB.